Molecular analysis of the Yeast Actin Cytoskeleton We will use biochemical and genetic approaches to study actin assembly in yeast. These studies will help to elucidate the molecular mechanisms responsible for cellular morphogenesis. The sophisticated classical and molecular genetics of the yeast Saccharomyces cerevisiae, together with there simplicity of the organism compared to higher cells, make it particularly tractable for these studies. These specific questions will be addressed: 1. How does actin assemble? The assembly properties of wild type and mutant actins will be determined in vitro to provide functional tests of the molecular model of the actin filament. In addition, phenotypic analysis of a full spectrum of actin mutants will provide a better definition of there in vivo roles of actin. 2. What are the roles of actin-binding proteins? The two yeast actin-binding proteins encoded by the SAC6 and ABP1 genes will be studied biochemically and genetically to gain an understanding of these proteins at a level not yet achieved for actin-binding proteins in previous studies using genetically intractable organisms. The function an ABP1 protein domain homologous to tyrosine kinase proto-oncogene regulatory domains will be explored in detail. 3. How is the organization of the actin cytoskeleton controlled spatially and temporally? Biochemical and genetic approaches will be used to identify structural proteins that bind to and organize the actin cytoskeleton. Classical genetic approaches will be used to identify the proteins that transduce regulatory signals to the actin cytoskeleton, and second messenger molecules that can affect actin-binding protein activities will be identified using biochemical assays. In total, these studies will address central questions about the function of the actin cytoskeleton in vivo and about how the cytoskeleton is organized; the disposition of the cytoskeleton within the cell is central to its function. What is learned from these studies is likely to apply to higher cells where defective actin cytoskeleton function is implicated in pathological conditions such as muscular dystrophy and neoplastic transformation.